A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling.

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  • Additional Information
    • Source:
      Publisher: Elsevier/North-Holland Biomedical Press Country of Publication: Netherlands NLM ID: 7905558 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1872-678X (Electronic) Linking ISSN: 01650270 NLM ISO Abbreviation: J Neurosci Methods Subsets: MEDLINE
    • Publication Information:
      Original Publication: Amsterdam, Elsevier/North-Holland Biomedical Press.
    • Subject Terms:
      Antimetabolites* ; Bromodeoxyuridine* ; Neurogenesis*; DNA/*metabolism ; Immunohistochemistry/*methods ; Neural Stem Cells/*metabolism ; Oligodendroglia/*metabolism ; Staining and Labeling/*methods ; Thymidine/*analysis; Animals ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Nucleic Acid Denaturation
    • Abstract:
      The typical immunohistochemistry technique used to reveal 5-bromo-2'-deoxyuridine (BrdU) incorporation requires denaturation of the DNA by heat and acid to permeabilize the cell nucleus. This treatment can damage tissue and reduce the antigenicity of several proteins, which then leads to weak immunostaining and/or false negatives. We show that an overnight post-fixation step following immunohistochemistry for antigens of interest protects immunostaining during the acid/heat denaturation treatment for subsequent BrdU staining. We used this technique to study the differentiation of recently divided oligodendrocyte progenitor cells in NG2CreER:EYFP reporter mice. We used a GFP anti-EYFP antibody to maximize visualization of the EYFP-containing oligodendrocyte progenitor cells, Olig1, and GST-pi to confirm the cell phenotype. Immunostaining for GFP, Olig1, and GST-pi is reduced by DNA denaturation. We found that incorporating a post-fixation step after double immunostaining for GFP/Olig1 and GFP/GST-pi prior to DNA denaturation prevented the fading and false negatives associated with this treatment. This simple addition to BrdU immunohistochemistry protocols extends the range of proteins that can be detected in combination with BrdU, along with the number of antibodies that can be used successfully in the study of cell proliferation.
      (Copyright © 2015 Elsevier B.V. All rights reserved.)
    • Contributed Indexing:
      Keywords: BrdU; Denaturation; Gliogenesis; Immunohistochemistry; Neurogenesis
    • Accession Number:
      0 (Antimetabolites)
      9007-49-2 (DNA)
      G34N38R2N1 (Bromodeoxyuridine)
      VC2W18DGKR (Thymidine)
    • Publication Date:
      Date Created: 20151202 Date Completed: 20161213 Latest Revision: 20170801
    • Publication Date:
      20221213
    • Accession Number:
      10.1016/j.jneumeth.2015.11.006
    • Accession Number:
      26620201